EVALUATION OF PROTECTIVE EFFICACY OF FORMALIN INACTIVATED KYASANUR FOREST DISEASE VACCINE IN MICE
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Date
2022
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KARNATAKA VETERINARY, ANIMAL AND FISHERIES SCIENCES UNIVERSITY, BIDAR.
Abstract
The present study was undertaken with the objectives of evaluating safety and
efficacy of Kyasanur Forest Disease (KFD) vaccine inactivated with different
concentration of formalin in mice, quantification of Kyasanur Forest Disease virus (KFDV)
by Real time PCR and its comparison with in-vivo titration in mice and to study the
sequential experimental pathology of KFDV in mouse brain. In this study, KFD vaccine
prepared in Chicken Embryo Fibroblast was inactivated with 0.04%, 0.06%, and 0.08 %
concentrations of formalin. KFD vaccine inactivated with 0.04% and 0.06 % formalin
failed the safety test as mice inoculated with the vaccine developed disease symptoms
and/or death. KFD vaccine inactivated with 0.08% formalin passed the safety test since
none of the mice inoculated with this vaccine showed symptoms and/or death. 0.08%
formalin inactivated KFDV vaccine passed the potency test in mice with log protective
index of was 5.678. Since the formalin content is relatively lower (0.08%) than in the
currently available KFD vaccine (0.1%), this should induce no or lesser reactions of
pain/swelling at the site of inoculation, which may increase the vaccine acceptance and
vaccination coverage. The real time PCR on individual harvests yielded CT values of less
than 20 on all five harvests (H1 to H5). The real time PCR on tenfold dilutions of the pooled
harvests yielded CT values of 22.05, 24.13, 26.09, 29.34, 31.18, 34.02, 0.0 and 0.0 for
dilutions of 10-1, 10-2,10-3, 10-4, 10-5, 10-6, 10-7 and 10-8 respectively. Inoculations of these
tenfold dilutions in mice by conventional mice inoculation test revealed that the titre
(MLD50) of the virus of in the pooled harvest was at 10-6. Based on these findings, it could
be concluded that when the CT value of individual vaccine harvests in real time PCR is
less than 20, the KFD vaccine will have sufficient viral particles to pass the potency test.
Real time PCR on tenfold dilutions of the vaccine harvests indicated that the 1MLD50 of
the vaccine lies in the tenfold dilution that yields CT values between 31 to 34. The
sequential pathology of KFD virus in mice brain by intra cerebrally inoculating 1MLD50
of the virus showed symptoms of dullness, hunched back appearance, weakness, sluggish
movements with indications of hind quarter paralysis on day 4 pi. These symptoms got
aggravated with complete paralysis of hind quarters, inability to move followed by death
on 5th and 6th dpi. Microscopically, brain showed apoptosis of neurons, perivascular
cuffing, gliosis, congestion, neuropil vacuolation, meningitis, degeneration, and necrotic
neurons. Cerebral cortex, cerebellum and hippocampus that control the motor neuron
activities and muscle tone were primarily affected. Apoptosis of neurons was a consistent
and important brain lesion noticed in KFD infected mice. Present study is the first report
on apoptosis of neurons due to KFD virus infection in mice.